Epidemiological trends indicate that the incidence of abuse and overdose of the highly addictive opioid heroin have more than tripled in recent years. However, effective treatment options are lacking and this is reflected in the high rates of opioid relapse and overdose. At the center of this issue lies the ability of drug cravings to persist and even strengthen long after opioid detoxification. The behavioral mechanics of this incubation of craving effect have become well-characterized in recent years, but more molecular insight is required to identify druggable targets within the brain that sustain drug seeking after prolonged abstinence. To that end, the goal of this project is to identify mechanisms that sustain strong drug seeking, guided by the hypothesis that microRNA (miRNA)-mediated translational mechanisms contribute to continued heroin seeking after prolonged abstinence. While their role in opioid seeking has not been described, miRNAs are an ideal focus for this study because each miRNA can target hundreds of genes, giving a single miRNA a high degree of mechanistic flexibility and a wide genomic range, capable of orchestrating complex behaviors. Indeed, limited exploration into their role in addiction has yielded exciting and promising results, such a regulation by opioid agonists and modification of alcohol and cocaine seeking. To address the goal of the project, the mentored phase of the grant will be used to gain training in heroin self-administration, protein sequencing and bioinformatic tools to identify miRNA-protein target interactions. At present, the candidate is studying the role of miRNAs in traumatic memory storage in a mouse model of post-traumatic stress disorder (PTSD) and in preliminary data employed small RNA sequencing (miRNA-Seq) to measure lasting miRNA expression (>30 days) after a traumatic learning experience. During the mentored K99 period, the scope of the current project will be extended to perform proteomic analysis and align it with the existing miRNA-Seq dataset, enabling the identification of miRNA pathways that will be functionally tested for their support of traumatic stress memories in the PTSD model. In the R00 period, the candidate will then apply the acquired skills and knowledge of miRNAs and opioid pharmacology to study miRNA mechanisms in the incubation of heroin craving using a rodent model of heroin self-administration. Focusing on two brain regions involved in the incubation of opioid craving, the central amygdala and orbitofrontal cortex, expression of miRNAs and their pathways will be manipulated in vivo to functionally identify miRNA mechanisms that sustain drug seeking after 30 days of abstinence from heroin. Completion of this project will identify new mechanisms of opioid seeking in the drug- free brain, which represents a critical step towards preventing substance use relapse.